Virendra N. Pandey

Identification of cellular factors associated with the 3' nontranslated region of the hepatitis C virus genome

Chronic infection by hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remains largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3′-nontranslated region (3′-NTR) of the (+) strand HCV genome are essential for initiation of (−) strand synthesis. The RNA signals within the highly conserved 3′-NTR may be the site for recruiting cellular factors that mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this report, we demonstrate that an RNA affinity capture system developed in our laboratory used in conjunction with LC/MS/MS allowed us to positively identify more than 70 cellular proteins that interact with the 3′-NTR (+) of HCV. Binding of these cellular proteins was not competed out by a 10-fold excess of nonspecific competitor RNA. With few exceptions, all of the identified cellular proteins are RNA-binding proteins whose reported cellular functions provide unique insights into host cell-virus interactions and possible mechanisms influencing HCV replication and HCV-associated pathogenesis. Small interfering RNA-mediated silencing of selected 3′-NTR-binding proteins in an HCV replicon cell line reduced replicon RNA to undetectable levels, suggesting important roles for these cellular factors in HCV replication.

Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

ABSTRACT The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent.

The FUSE Binding Protein Is a Cellular Factor Required for Efficient Replication of Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma and one of the primary indications for liver transplantation. The molecular mechanisms underlying the actions of host factors in HCV replication remain poorly defined. FUSE (far upstream element of the c-myc proto-oncogene) binding protein (FBP) is a cellular factor that we have identified as a binder of HCV 3′ nontranslated region (3′NTR). Mapping of the binding site showed that FBP specifically interacts with the poly(U) tract within the poly(U/UC) region of the 3′NTR. Silencing of FBP expression by small interfering RNA in cells carrying HCV subgenomic replicons severely reduced viral replication, while overexpression of FBP significantly enhanced viral replication. We confirmed these observations by an in vitro HCV replication assay in the cell-free replicative lysate, which suggested that there is a direct correlation between the cellular FBP level and HCV replication. FBP immunoprecipitation coprecipitated HCV nonstructural protein 5A (NS5A), indicating that FBP interacts with HCV NS5A, which is known to function as a link between HCV translation and replication. Although FBP is mainly localized in the nucleus, we found that in MH14 cells a significant level of this protein is colocalized with NS5A in the cytosol, a site of HCV replication. While the mechanism of FBP involvement in HCV replication is yet to be delineated, our findings suggest that it may be an important regulatory component that is essential for efficient replication of HCV.

Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides

The transactivator responsive region (TAR) present in the 5′-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA–protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNATAR) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418–428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNATAR–peptide conjugates where uptake of the PNATAR–penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC50 values in the range of 28–37 nM while IC50 for inhibition of HIV-1 replication was lowest with PNATAR–transportan-27 (0.4 μM) followed by PNATAR–tat (0.72 μM) and PNATAR–penetratin (0.8 μM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.

Identification and evaluation of anti Hepatitis C Virus phytochemicals from Eclipta alba

ETHNOPHARMACOLOGICAL RELEVANCE Eclipta alba, traditionally known as bhringraj, has been used in Ayurvedic medicine for more than 1000 years in India. It is used for the treatment of infective hepatitis, liver cirrhosis, liver enlargement and other ailments of liver and gall bladder in India. The aim of this study was to evaluate anti-hepatitis C virus activity present in the Eclipta alba extract, perform bioassay based fractionation and identify anti-HCV phytochemicals from the active fractions. MATERIALS AND METHODS Identification of active compounds was performed by bio-activity guided fractionation approach. Active isolates were separated by the combination of silica gel chromatography and preparative scale reverse phase HPLC. Eclipta alba extract and its isolates were examined for their ability to inhibit HCV replicase (HCV NS5B) activity in vitro and HCV replication in a cell culture system carrying replicating HCV subgenomic RNA replicon. The purified isolates were also examined for their binding affinity to HCV replicase by fluorescence quenching and their cytotoxicity by MTT assay. RESULTS Eclipta alba extract strongly inhibited RNA dependent RNA polymerase (RdRp) activity of HCV replicase in vitro. In cell culture system, it effectively inhibited HCV replication which resulted in reduced HCV RNA titer and translation level of viral proteins. Bioassay-based fractionations of the extracts and purification of anti-HCV phytochemicals present in the active fractions have identified three compounds, wedelolactone, luteolin, and apigenin. These compounds exhibited dose dependent inhibition of HCV replicase in vitro, and anti-HCV replication activity in the cell culture system CONCLUSION Eclipta alba extract and phytochemicals isolated from active fractions display anti-HCV activity in vitro and in cell culture system. The standardized Eclipta alba extract or its isolates can be used as an effective alternative and complementary treatment against HCV.

Pharmacokinetic Analysis of Polyamide Nucleic-Acid-Cell Penetrating Peptide Conjugates Targeted against HIV-1 Transactivation Response Element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.

Insertion of a small peptide of six amino acids into the β7–β8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities

BackgroundHIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the β7-β8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme.ResultsThe polymerase and the RNase H activities of HIV-1 RT carrying insertion in the β7-β8 loop of both the subunits (p66INS/p51INS) were severely impaired with substantial loss of DNA binding ability. These enzymatic activities were restored when the mutant p66INS subunit was dimerized with the wild type p51. Glycerol gradient sedimentation analysis revealed that the mutant p51INS subunit was unable to form stable dimer either with the wild type p66 or mutant p66INS. Furthermore, the p66INS/p66INS mutant sedimented as a monomeric species, suggesting its inability to form stable homodimer.ConclusionThe data presented herein indicates that any perturbation in the β7-β8 loop of the p51 subunit of HIV-1 RT affects the dimerization process resulting in substantial loss of DNA binding ability and catalytic function of the enzyme.

STAT3 Inhibition in Prostate and Pancreatic Cancer Lines by STAT3 Binding Sequence Oligonucleotides: Differential Activity Between 5′ and 3′ Ends

Signal transducers and activators of transcription (STAT) were originally discovered as components of signal transduction pathways. Persistent aberrant activation of STAT3 is a feature of many malignancies including prostate cancer and pancreatic cancer. One consequence of persistently activated STAT3 in malignant cells is that they depend on it for survival; thus, STAT3 is an excellent molecular target for therapy. Previously, we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. Here, we report that authentic STAT3 binding sequences, identified from published literature, were more effective for inducing apoptosis in prostate cancer cells and pancreatic cancer cells than was oligonucleotide 13410. Moreover, the authentic STAT3 binding sequences showed differing efficacies in the malignant cell lines depending on whether the canonical STAT3 binding sequence was truncated at the 5′ or the 3′ end. Finally, expression of one STAT3-regulated gene was decreased following treatment, suggesting that STAT3 may regulate the same set of genes in the two types of cancer. We conclude that truncating the 5′ end left intact enough of the canonical STAT3 binding site for effective hybridization to the genome, whereas truncation of the 3′ end, which is outside the canonical binding site, may have affected binding of required cofactors essential for STAT3 activity, thereby reducing the capacity of this modified oligonucleotide to induce apoptosis. Additional experiments to answer this hypothesis are under way. [Mol Cancer Ther 2008;7(6):1543–50]

A peptide nucleic acid-aminosugar conjugate targeting transactivation response element of HIV-1 RNA genome shows a high bioavailability in human cells and strongly inhibits tat-mediated transactivation of HIV-1 transcription.

The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16-mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose, we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic conditions, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment because cotreatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in a cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application.

Single Acute-Dose and Repeat-Doses Toxicity of anti-HIV-1 PNATAR–Penetratin Conjugate after Intraperitoneal Administration to Mice

Polyamide (peptide) nucleic acids conjugated with membrane-penetrating peptide are potential antisense therapeutic agents because of their unique chemical properties, high target specificity, and efficient cellular uptake. However, studies of their potential toxicity in animal models are lacking. In this study, we evaluated the toxicity of the response of Balb/C mice to anti-HIV-1 PNA TAR-penetratin conjugate targeted against the transactivation response (TAR) element of HIV-1 LTR. A single i.p. dose of 600 mg/kg of body weight was lethal, killing all mice within 72 hours. However, death did not occur after single doses of 100 and 300 mg/kg, although all mice experienced initial and transitory diarrhea and loss of agility. Repeated daily doses of 10, 30, and 100 mg/kg were well tolerated by mice during 8 days of treatment, although daily doses of 100 mg/kg caused diarrhea during the first 4 days of treatment. During 8 weeks of follow-up, mice fully recuperated. Serositis was observed in the spleens, livers, and kidneys at the ninth day of treatment, but not after the follow-up period. Necropsies, clinical chemistry studies, and hematological parameters demonstrated normal function of the major organs and no irreversible damage to the mice. These observations indicate that the PNA-peptide conjugate would be nontoxic at probable therapeutic doses and thus support its therapeutic potential as an antisense drug.